Enzymatic synthesis of sulfated polysaccharides

ABSTRACT

A method of sulfating a polysaccharide is provided. The method can include providing a reaction mixture comprising at least one O-sulfotransferase (OST) enzyme and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); incubating a polysaccharide substrate with the reaction mixture, wherein production of the sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate (PAP); and providing a reaction condition which modifies PAP to reduce an inhibitory effect of PAP on the polysaccharide sulfation.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/680,392, filed May 12, 2005; the disclosure ofwhich is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST

The presently disclosed subject matter was made with U.S. Governmentsupport under Grant No. AI50050 awarded by the National Institutes ofHealth. Thus, the U.S. Government has certain rights in the presentlydisclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter relates generally to methods ofsulfating polysaccharides. In particular, the presently disclosedsubject matter relates to methods of sulfating polysaccharides usingO-sulfotransferases, which includes a reaction condition that reducespotential inhibitory effects from sulfur donor byproducts.

BACKGROUND

Heparan sulfate (HS) is a ubiquitous component of the cell surface andextracellular matrix. It regulates a wide range of physiologic andpathophysiologic functions, including embryonic development and bloodcoagulation, and can facilitate viral infection (Esko and Selleck (2002)Annu. Rev. Biochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 22,1-25). HS exerts its biological effects by interacting with the specificproteins involved in a given process (Capila and Lindhardt (2002) Angew.Chem. Int. Ed. 41, 390-412). HS is a highly charged polysaccharideconsisting of 1→4-linked glucosamine and glucuronic/iduronic acid unitsthat contain both N- and O-sulfo groups. Unique saccharide sequenceswithin HS determine the specificity of the binding of HS to its targetproteins (Linhardt (2003) J. Med. Chem. 46, 2551-2564). Heparin, aspecialized form of HS, is a commonly used anticoagulant drug. Thus, newmethods for the synthesis of heparin and HS attract considerableinterest for those developing anticoagulant and other HS-related drugshaving improved pharmacological effects.

Chemical synthesis has been the major route to obtain structurallydefined heparin and HS oligosaccharides (Petitou and van Boeckel (2004)Angew. Chem. Int. Ed. 43, 3118-3133). One example of a chemicallysynthesized HS oligosaccharide is a synthetic pentasaccharide havingantithrombin-binding properties marketed in the United States under thetrade name ARIXTRA® (GlaxoSmithKline, Middlesex, United Kingdom).ARIXTRA® is a specific factor Xa inhibitor that is used clinically toprevent venous thromboembolic incidents during surgery.

Unfortunately, the total synthesis of heparin and HS oligosaccharides,larger than pentasaccharides, is difficult. HS analogues with 14saccharide units inhibit the activity of thrombin, but these syntheticanalogues are simplified hybrid molecules of HS oligosaccharides andhighly sulfated glucose units (Petitou et al. (1999) Nature 398,417-422; Dementiev et al. (2004) Nat. Struct. Biol. 11, 867-863) and arenot the naturally occurring structures. Although the pursuit for thechemical synthesis of heparin and HS oligosaccharides (Avci et al.(2003) Curr. Pharm. Des. 9, 2323-2335) continues, it has become clearthat chemical synthesis alone is currently incapable of generating mostlarger oligosaccharide structures. Thus, the application of HSbiosynthetic enzymes for generating large heparin and HSoligosaccharides with desired biological activities offers a promisingalternative approach.

Six classes of enzymes are involved in HS biosynthesis. HS is initiallysynthesized as a copolymer of D-glucuronic acid and N-acetylglucosamine(GlcNAc) through the action of D-glucuronyl andN-acetyl-D-glucosaminyltransferase (Lindahl et al. (1998) J. Biol. Chem.273, 24979-24982). Next, a series of modifications take place, includingN-deacetylation and N-sulfation (carried out byN-deacetylase/N-sulfotransferase) of the glucosamine residue to formN-sulfoglucosamine (GlcNS), C₅ epimerization of glucuronic acid (carriedout by epimerase) to form L-iduronic acid (IdoUA), 2-O-sulfation ofIdoUA (carried out by 2-O-sulfotransferase (2-OST)), 6-O-sulfation ofglucosamine (carried out by 6-O-sulfotransferase (6-OST)), and3-O-sulfation of glucosamine (carried out by 3-O-sulfotransferase(3-OST)) (Sasisekharan et al. (2002) Nat. Rev. Cancer 2, 521-528). Thereactions catalyzed by 2-OST, 6-OST, and 3-OST are shown in FIG. 1A.

Enzymes “in the pathway” for HS biosynthesis have been cloned andexpressed, and have been employed in the synthesis of HSpolysaccharides. Kuberan and Rosenberg (Balagurunathan et al. (2003)Nat. Biotechnol. 21, 1343-1346; Kuberan et al. (2003) J. Am. Chem. Soc.125, 12424-12425; Balagurunathan et al. (2003) J. Biol. Chem. 278,52613-52621) utilized these enzymes to synthesize an HS containingantithrombin binding sites with anticoagulant activity. Although thisapproach demonstrated for the first time the feasibility of enzymaticsynthesis of HS, only about 1 μg of product was generated, makingextensive structural characterization and biological studies impossible.Recently, Lindahl and colleagues reported an alternative chemoenzymaticapproach for the synthesis of anticoagulant heparin from heparosan, theE. coli K5 capsular polysaccharide (Lindahl et al. (2005) J. Med. Chem.48, 349-352). This method utilized the C₅ epimerase to convertD-glucuronic acid to IdoUA, followed by the chemical persulfation andfinally selective desulfation. Although this approach affordedapproximately 5 g of a heparin-like polysaccharide with anticoagulantactivity, unnatural saccharide units, such as 3-O-sulfo-D-glucuronicacid, were present in their product. This suggested a limitation in theselectivity of chemical sulfation/desulfation in HS synthesis. Further,the OST catalyzed sulfation reaction utilizes 3′-phosphoadenosine5′-phosphosulfate (PAPS) as the sulfur donor, producing adenosine3′,5′-diphosphate (PAP). PAP can compete with PAPS for OST binding,which can result in inhibition of the sulfation reaction over time asPAP concentration increases in the reaction mixture.

Thus, developing an effective and highly selective approach forO-sulfation of polysaccharides remains an unmet need in the art for thelarge scale synthesis of HS.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments of the presently disclosed subject matter, a methodof sulfating a polysaccharide is provided, comprising (a) providing areaction mixture comprising at least one O-sulfotransferase (OST) enzymeand 3′-phosphoadenosine 5′-phosphosulfate (PAPS); (b) incubating apolysaccharide substrate with the reaction mixture, wherein productionof the sulfated polysaccharide from the polysaccharide substrate iscatalyzed by the OST enzyme with a conversion of the PAPS to adenosine3′,5′-diphosphate (PAP); and (c) providing a reaction condition whichmodifies PAP to reduce an inhibitory effect of PAP on the polysaccharidesulfation. In some embodiments, providing the reaction conditioncomprises providing a PAPS regeneration system comprising a PAPSregenerating enzyme and a sulfur donor compound, wherein the PAPSregenerating enzyme catalyzes regeneration of the PAPS from the PAPutilizing the sulfur donor compound as a substrate. In some embodiments,the PAPS regenerating enzyme is an arylsulfotransferase, such as forexample AST-IV. In some embodiments, the sulfur donor compound is anaryl sulfate compound, such as for example p-nitrophenol sulfate (PNPS).In some embodiments, providing the reaction condition comprisesproviding a phosphatase enzyme, wherein the phosphatase enzyme modifiesthe PAP.

In some embodiments of the presently disclosed subject matter, a methodof sulfating a polysaccharide is provided, comprising (a) providing areaction mixture comprising PAP, a PAPS regenerating enzyme and a sulfurdonor compound; (b) incubating the reaction mixture for a time periodsufficient to catalyze the production of PAPS from the PAP by the PAPSregenerating enzyme utilizing the sulfur donor compound as a substrate,such as for example a time period from about 1 minute to about 30minutes; and (c) incubating a polysaccharide substrate and at least oneOST enzyme with the reaction mixture, wherein production of a sulfatedpolysaccharide from the polysaccharide substrate is catalyzed by the OSTenzyme with a conversion of the PAPS to PAP and wherein the PAPSregenerating enzyme catalyzes regeneration of the PAPS from the PAPutilizing the sulfur donor compound as a substrate. In some embodiments,the PAPS regenerating enzyme is an arylsulfotransferase, such as forexample AST-IV. In some embodiments, the donor compound is an arylsulfate compound, such as for example PNPS.

In some embodiments of the methods for sulfating polysaccharides, the atleast one OST enzyme is selected from the group consisting of 2-OST,3-OST-1, 3-OST-3, 6-OST, and combinations thereof. In some embodiments,the at least one OST enzyme is a recombinant OST enzyme, which is, insome embodiments, produced in a bacterial expression system. Further, insome embodiments, the OST enzyme is a fusion protein, such as forexample a maltose-binding protein (MBP)-2-OST fusion protein or aMBP-6-OST fusion protein. In some embodiments, the OST enzyme isimmobilized on a substrate, such as for example an agarose bead.

In some embodiments of the methods for sulfating polysaccharides, thepolysaccharide substrate is a chemically desulfated N-sulfated (CDSNS)heparin. In some embodiments, the polysaccharide substrate is partiallysulfated prior to reaction mixture incubation. In some embodiments, thesulfated polysaccharide is a glycosaminoglycan (GAG), such as forexample a heparan sulfate (HS). In some embodiments, the sulfatedpolysaccharide is an HS that is an anticoagulant-active HS, anantithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, aherpes simplex virus envelope glycoprotein D-binding HS, or has acombination of these properties.

In some embodiments of the presently disclosed subject matter, a kit forsulfating a polysaccharide is provided. In some embodiments, the kitcomprises at least one OST enzyme and a reagent which modifies PAP toreduce an inhibitory effect of PAP on polysaccharide sulfation. In someembodiments, the kit further comprising instructions for sulfating apolysaccharide. In some embodiments, the at least one OST enzyme iscontained within a first container and the reagent is contained within asecond container. In some embodiments, the at least one OST enzyme isselected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST,and combinations thereof. Further, in some embodiments, the OST enzymeis a recombinant OST enzyme, such as for example a recombinant OSTenzyme produced in a bacterial expression system. In some embodiments,the OST enzyme is a fusion protein, such as for example amaltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusionprotein. In some embodiments, the OST enzyme is immobilized on asubstrate, such as for example an agarose bead.

In some embodiments of the kit, the reagent comprises a PAPSregeneration system comprising a PAPS regenerating enzyme (e.g., anarylsulfotransferase, such as AST-IV) and a sulfur donor compound (e.g.,an aryl sulfate compound, such as PNPS. In other embodiments of the kit,the reagent comprises a phosphatase enzyme.

Accordingly, it is an object of the presently disclosed subject matterto provide for the enzymatic synthesis of sulfated polysaccharides. Thisobject is achieved in whole or in part by the presently disclosedsubject matter.

An object of the presently disclosed subject matter having been statedabove, other objects and advantages will become apparent to those ofordinary skill in the art after a study of the following description ofthe presently disclosed subject matter and non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic drawings showing synthesis of sulfatedpolysaccharides coupled with a PAPS enzymatic regeneration system. FIG.1A shows the stepwise enzymatic synthesis of sulfated polysaccharidesusing HS O-sulfotransferases. The description of intermediatepolysaccharides is disclosed in the Examples herein below. Compounds 4aand 4b were prepared by inverting the order of sulfation steps. 4a wasprepared by incubating compound 1 with 2-OST followed by 6-OST, whereas4b was prepared by incubating compound 1 with 6-OST followed by 2-OST.FIG. 1B shows the reaction catalyzed by arylsulfotransferase IV (AST-IV)to generate PAPS. R represents —H or —SO₃.

FIG. 2 is a graph showing activities of recycled immobilizedsulfotransferases. The immobilized enzymes were utilized in multiplecycles. The activities of the immobilized enzymes after each cycle weredetermined as described in the Examples herein below. A total of 10cycles was conducted. 2-OST (•); 6-OST (∘); 3-OST-1 (▾); and 3-OST-3(Δ).

FIGS. 3A-3E are RPIP-HPLC chromatograms of the disaccharide analysis ofsynthesized polysaccharides. The synthesized polysaccharides weredigested with a mixture of heparinases, including heparin lyase I, II,III, and heparinase IV. The resultant disaccharides were purified byBioGel P-2 and resolved on RPIP-HPLC. FIGS. 3A-3E show the chromatogramsof the disaccharide analysis of compounds 1, 2, 3, 4a, and 4b, from FIG.1A respectively. The numbers above peaks indicate the eluted positionsof authentic disaccharide standards, where 1 represents ΔUA-GlcNAc, 2represents ΔUA-GlcNS, 3 represents ΔUA-GlcNS6S, 4 representsΔUA2S-GlcNS, and 5 represents ΔUA2S-GlcNS6S. * marks the eluted positionof ΔUA2S-GlcNAc. The quantity of ΔUA2S-GlcNAc not determined.

FIGS. 4A and 4B are graphs showing the effect of the synthesizedpolysaccharides on FGF-2-dependent BaF3 FGFR1c cell proliferation. FIG.4A, BaF3 FGFR1c cells were seeded in 96-well plates as described with 2nM FGF2 for control and 2 nM FGF2 plus a 1 μg/ml concentration of thefollowing compounds: heparin, 1, 2, 3, 4a, and 4b (FIG. 1A). FIG. 4Bshows dose-response curves of heparin, 4a, and 4b for their activitiesin stimulating cell proliferation. Cells were cultured for 40 hours (h),followed by incubating in the media containing [³H]thymidine for 4 h.The cellular proliferation was determined by [³H]thymidine incorporationinto the DNA. heparin (•); 4a (∘); and 4b (▾). Data are mean±range ofduplicates.

DETAILED DESCRIPTION

The details of one or more embodiments of the presently disclosedsubject matter are set forth in the accompanying description below.Other features, objects, and advantages of the presently disclosedsubject matter will be apparent from the detailed description, and fromthe claims. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. Some of the polynucleotide and polypeptide sequences disclosedherein are cross-referenced to GENBANK® accession numbers. The sequencescross-referenced in the GENBANK® database are expressly incorporated byreference as are equivalent and related sequences present in GENBANK® orother public databases. Also expressly incorporated herein by referenceare all annotations present in the GENBANK® database associated with thesequences disclosed herein. In case of conflict, the presentspecification, including definitions, will control.

I. General Considerations

Heparan sulfates (HSs) are highly sulfated polysaccharides, present onthe surface of mammalian cells and in the extracellular matrix in largequantities. HS is a highly charged polysaccharide consisting of1→4-linked glucosamine and glucuronic/iduronic acid units that containboth N- and O-sulfo groups. Heparin, a specialized form of HS, is acommonly used anticoagulant drug. Thus, “heparan sulfate”, as usedherein, includes heparin.

HSs play critical roles in a variety of important biological processes,including assisting viral infection, regulating blood coagulation andembryonic development, suppressing tumor growth, and controlling theeating behavior of test subjects by interacting with specific regulatoryproteins (Liu, J., and Thorp, S. C. (2002) Med. Res. Rev. 22:1-25;Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070;Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68:729-777; Alexander,C. M., et al., (2000) Nat. Genet. 25:329-332; Reizes, O., et al., (2001)Cell 106:105-116). The unique sequences determine to which specificproteins HSs bind, thereby regulating biological processes.

The biosynthesis of HS occurs in the Golgi apparatus. It is initiallysynthesized as a copolymer of glucuronic acid and N-acetylatedglucosamine by D-glucuronyl and N-acetyl-D-glucosaminyltransferase,followed by various modifications (Lindahl, U., et al., (1998) J. Biol.Chem. 273:24979-24982). These modifications include N-deacetylation andN-sulfation of glucosamine, C₅ epimerization of glucuronic acid to formiduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid,as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Severalenzymes that are responsible for the biosynthesis of HS have been clonedand characterized (Esko, J. D., and Lindahl, U. (2001) J. Clin. Invest.108:169-173).

The expression levels of various HS biosynthetic enzyme isoformscontribute to the synthesis of specific saccharide sequences in specifictissues. HS N-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and6-O-sulfotransferase are present in multiple isoforms. Each isoform isbelieved to recognize a saccharide sequence around the modification sitein order to generate a specific sulfated saccharide sequence (Liu, J.,et al., (1999) J. Biol. Chem. 274:5185-5192; Aikawa, J.-I., et al.,(2001) J. Biol. Chem. 276:5876-5882; Habuchi, H., et al., (2000) J.Biol. Chem. 275:2859-2868). For instance, HS D-glucosaminyl3-O-sulfotransferase (3-OST) isoforms generate 3-O-sulfated glucosamineresidues that are linked to different sulfated uronic acid residues.3-OST isoform 1 (3-OST-1) transfers sulfate to the 3-OH position of anN-sulfated glucosamine residue that is linked to a glucuronic acidresidue at the nonreducing end (GlcUA-GlcNS±6S). However, 3-OST isoform3 (3-OST-3) transfers sulfate to the 3-OH position of an N-unsubstitutedglucosamine residue that is linked to a 2-O-sulfated iduronic acid atthe nonreducing end (IdoUA2S-GlcNH₂±6S) (Liu, J., et al., (1999) J.Biol. Chem. 274:38155-38162). The difference in the substratespecificity of 3-OSTs results in distinct biological functions. Forexample, the HS modified by 3-OST-1 binds to antithrombin (AT) andpossesses anticoagulant activity (Liu, J., et al., (1996) J. Biol. Chem.271:27072-27082). However, the HS modified by 3-OST-3 (3-OST-3A and3-OST-3B) binds to glycoprotein D (gD) of herpes simplex virus, type 1,(HSV-1) thus mediating viral entry (Shukla, D., et al., (1999) Cell99:13-22).

The HS- and heparin-regulated anticoagulation mechanisms have beenstudied extensively. It is now known that HS, including heparin,interact with AT, a serine protease inhibitor, to inhibit the activitiesof thrombin and factor Xa in the blood coagulation cascade (Rosenberg,R. D., et al., (1997) J. Clin. Invest 99:2062-2070).Anticoagulant-active HS(HS^(act)) and heparin contain one or multipleAT-binding sites per polysaccharide chain. This binding site contains aspecific pentasaccharide sequence with a structure of -GlcNS(orAc)6S-GlcUA-GlcNS3S(±6S)-IdoUA2S-GlcNS6S-. The 3-O-sulfation ofglucosamine for generating GlcNS3S(±6S) residue, which is carried out by3-OST-1, is an important modification for the synthesis of HS^(act)(Liu, J., et al., (1996) J. Biol. Chem. 271:27072-27082; Shworak, N. W.,et al., (1997) J. Biol. Chem. 272:28008-28019).

Cell surface HS also assists HSV-1 infection (WuDunn, D., and Spear, P.G. (1989) J. Virol. 63:52-58). One report (Shukla, D., et al., (1999)Cell 99:13-22) suggests that a specific 3-O-sulfated HS is involved inassisting HSV-1 entry. The 3-O-sulfated HS is generated by 3-OST-3 butnot by 3-OST-1. In addition, the 3-O-sulfated HS provides binding sitesfor HSV-1 envelope glycoprotein D, which is a key viral protein involvedin the entry of HSV-1 (Shukla, D., et al., (1999) Cell 99:13-22).Because 3-OST-3-modified HS is rarely found in HS from natural sources,the study suggests that HSV-1 recognizes a unique saccharide structure.Indeed, the result from the structural characterization of a gD-bindingoctasaccharide revealed that the octasaccharide possesses a specificsaccharide sequence (Liu, J., et al., (2002) J. Biol. Chem.277:33456-33467). In addition, the binding affinity of the 3-O-sulfatedHS for gD is about 2 μM (Shukla, D., et al., Cell 99:13-22). Thisaffinity is similar to that reported for the binding of gD to theprotein receptors, suggesting that HSV-1 utilizes both protein and HScell surface receptors to infect target cells (Willis, S. H., et al.,(1998) J. Virol. 72:5938-5947; Krummenacher, C., et al., (1999) J.Virol. 73:8127-8137). It is believed that the interaction between gD andthe 3-O-sulfated HS or the protein entry receptors somehow triggers thefusion between the virus and the cell in the presence of other viralenvelope proteins, including gB, gH, and gL (Shukla, D., and Spear, P.G. (2001) J. Clin. Invest. 108:503-510). A study of the co-crystalstructure of gD and herpes entry receptor HveA suggests that the bindingof HveA to gD induces conformational changes in gD (Carfi, A., et al.,(2001) Mol. Cell 8:169-179).

II. Methods of Sulfating Polysaccharides

The presently disclosed subject matter provides enzymatic methods forthe sulfation of multimilligram amounts of heparan sulfate havingparticular functions using sulfotransferases coupled with a system forreducing inhibitory effects from sulfur donor byproducts. In someembodiments, the system for reducing inhibitory byproducts comprises a3′-phosphoadenosine 5′-phosphosulfate regeneration system. In otherembodiments, the system comprises a phosphatase enzyme. By utilizing thepresently disclosed sulfation system and selecting appropriate enzymaticmodification steps, an inactive precursor polysaccharide can beenconverted to a heparan sulfate having desired biological properties.

In some embodiments, the presently disclosed subject matter employsrecombinant sulfotransferases. Because the recombinant sulfotransferasescan be recombinantly expressed in bacteria, and the disclosed methodscan use low cost sulfo donors, the presently disclosed subject mattercan be readily utilized to synthesize large quantities of biologicallyactive heparan sulfates while reducing the production of reactioninhibitory byproducts.

Two advantages provided by the presently disclosed subject matterfacilitate the large scale synthesis of HS. First, large amounts of allthe required HS sulfotransferases can be successfully recombinantlyexpressed in Escherichia coli. Second, the enzymatic sulfation reactionsare coupled with a system for reducing inhibitory effects from sulfurdonor byproducts (e.g., PAP) and reducing costs related to continuouslyproviding a supply of the sulfur donor PAPS. PAPS, a universal sulfatedonor and source of sulfate for all sulfotransferases, is a highlyexpensive and unstable molecule that has been an obstacle to thelarge-scale production of enzymatically sulfated products. The half-lifeof PAPS in aqueous solution at pH 8.0 is approximately 20 hours. Productinhibition by adenosine 3′,5′-diphosphate (PAP) has also been a limitingfactor to large-scale applications. For example, PAP inhibition ofhydroxysteroid sulfotransferase was determined to be K_(i)=14 μM (Marcuset al. (1980) Aial. Biochem. 107, 296). PAP has also been shown toinhibit the sulfotransferase NodST with a K_(i)=0.1 μM (Lin et al.,(1995) J. Am. Chem. Soc. 117, 8031). In some embodiments of thepresently disclosed subject matter, a PAPS regeneration system, such asthe system developed by Burkhart and colleagues (Burkart et al. (2000)J. Org. Chem. 65, 5565-5574, incorporated herein by reference), has beenmodified and adapted to be coupled to the enzymatic synthesis reactions.The PAPS regeneration system converts PAP into PAPS, thereby reducingaccumulation of inhibitory PAP in the reaction mixture and reducingproduction costs related to providing PAPS to drive the sulfationreaction. In other embodiments, phosphatase enzymes can be utilized tomodify PAP so that it no longer has binding affinity forsulfotransferases.

The presently disclosed sulfation system can be adapted to produce amultitude of HS molecules having varied biological activities byselecting appropriate sulfotransferases to include and by sequentiallycontrolling the addition of those sulfotransferases to the reactionsystem to facilitate appropriate timing of sulfations of thepolysaccharide template. For example, as disclosed herein, HS havingspecific biological activities can be synthesized utilizing thepresently disclosed methods, including anticoagulant HS, fibroblastgrowth factor-2-binding activity, herpes simplex virus glycoprotein D(gD)-binding HS, and fibroblast growth factor 2 (FGF2) receptor-bindingHS. Only two or three enzymatic steps are required for the synthesis ofeach of these biologically-active HS molecules (FIG. 1A). Thus, thepresently disclosed subject matter provides an efficient and effectivemethod for the large scale synthesis of a wide range of HS with specificactivity required. In addition, this method provides a model system tobetter understand the biosynthesis of HS.

In some embodiments of the presently disclosed subject matter, a methodof sulfating a polysaccharide is provided. In some embodiments, themethod comprises incubating a polysaccharide substrate to be sulfatedwith a reaction mixture that comprises at least one sulfotransferaseenzyme, such as for example an O-sulfotransferase (OST) enzyme, and asulfur donor, such as for example PAPS. Production of the sulfatedpolysaccharide from the polysaccharide substrate is catalyzed by the OSTenzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate(PAP). A reaction condition is further provided that modifies generatedPAP to reduce an inhibitory effect of PAP on the polysaccharidesulfation. For example, a PAPS regeneration system can be coupled withthe sulfation reaction to convert PAP into PAPS or phosphatases can beadded to the reaction mixture to modify PAP such that it does notcompete with PAPS for binding with OSTs.

In some embodiments, the polysaccharide substrate is a previouslyN,O-desulfated and re-N-sulfated polysaccharide, such as for example achemically desulfated N-sulfated (CDSNS) heparin. In other embodiments,the polysaccharide substrate is partially sulfated prior to reactionmixture incubation. For example, a CDSNS can be reacted with aparticular OST to produce a sulfated polysaccharide intermediate productthat can then be reacted with a different OST to further sulfate thepolysaccharide at different locations. This sequential process ofreacting the polysaccharide substrate with different OSTs can becontinued until a final polysaccharide is produced exhibiting desiredbiological activities (based, at least in part, on the sulfation patternof the polysaccharide). FIG. 1A schematically illustrates severalexemplary polysaccharide substrates sequentially reacted with differentOSTs to produce different intermediate and end products. For example,compound 1 can be reacted with 2-OST and then 6-OST to produce compound4a or compound 1 can be reacted with 6-OST and then 2-OST to producecompound 4b, each having fibroblast growth factor (FGF)-bindingactivity. Further, compounds 4a or 4b can be reacted with 3-OST-1 toproduce compound 5 having antithrombin-binding and anticoagulantactivities. Alternatively, compounds 4a or 4b can be reacted with3-OST-3 to produce compound 6 having herpes simplex virus (HSV) envelopeglycoprotein D (gD) binding activity. As such, the presently disclosedsubject matter provides for the production of HS compounds havingdifferent biological properties based on the selection and sequentialreaction of different OSTs with polysaccharide substrates. Thepolysaccharide substrate can be reacted with different OST enzymes byaddition of each enzyme sequentially to the same reaction mixture, orintermediate polysaccharide products can be purified from the reactionmixture after reaction of a particular OST and then reacted with adifferent OST. In some embodiments, depending on the desired finalproduct, different OST enzymes can be added to the reaction mixturesimultaneously.

In some embodiments the sulfated polysaccharide product can be aglycosaminoglycan (GAG). GAGs are the most abundantheteropolysaccharides in the body. These molecules are long unbranchedpolysaccharides containing a repeating disaccharide unit. Thedisaccharide units can contain either of two modified sugars:N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and auronic acid such as glucuronate or iduronate. GAGs are highly negativelycharged molecules, with extended conformation that imparts highviscosity to the solution. Along with the high viscosity of GAGs comeslow compressibility, which makes these molecules ideal for a lubricatingfluid in the joints. At the same time, their rigidity providesstructural integrity to cells and provides passageways between cells,allowing for cell migration. The specific GAGs of physiologicalsignificance are hyaluronic acid, dermatan sulfate, chondroitin sulfate,heparin, heparan sulfate (including heparin), and keratan sulfate. Thus,in some embodiments, the sulfated polysaccharide product is a HS. Insome embodiments, the sulfated polysaccharide product is ananticoagulant-active HS, an antithrombin-binding HS, an FGF-binding HS,and an HSV gD-binding HS.

II.A. Sulfotransferases

As previously noted, the presently disclosed subject matter utilizessulfotransferases, particularly O-sulfotransferases (OSTs), to sulfatepolysaccharides. Sulfotransferases comprise a family of enzymes thatcatalyze the transfer of a sulfonate or sulfuryl group (SO₃) from thecofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to an acceptormolecule. Even though it is more accurate to call these sulfonationreactions, the term sulfation is still widely used. Therefore, the term“sulfation” as used herein refers to a transfer of a sulfonate orsulfuryl group from one molecule to another.

Sulfotransferases mediate sulfation of different classes of substratessuch as carbohydrates, oligosaccharides, peptides, proteins, flavonoids,and steroids for a variety of biological functions including signalingand modulation of receptor binding (Bowman et al., (1999) Chem. Biol. 6,R9-R22; and Falany (1997) FASEB J. 11, 1-2). Within the past few years,many new sulfotransferases have been identified and cloned (Aikawa etal., (1999) J. Biol. Chem. 274, 2690; Dooley (1998) Chemico-Biol.Interact. 109, 29; Fukuta et al. (1998) Biochim. Biophys. Act. 1399, 57;Habuchi et al., (1998) J. Biol. Chem. 273, 9208; Mazany et al., (1998)Biochim. Biophys. Act. 1407, 92; Nastuk et al. (1998) J. Neuroscience18, 7167; Ong et al., (1998) J. Biol. Chem. 273, 5190; Ouyang et al.,(1998) J. Biol. Chem. 273, 24770; Saeki et al. (1998) J. Biochem. 124,55; Uchimura et al. (1998) J. Biol. Chem. 273, 22577; and Yoshinari etal., (1998) J. Biochem. 123, 740).

As used herein, the term “O-sulfotransferase (OST)” includespolypeptides and nucleic acids encoding HS O-sulfotransferases, such asfor example “2-OST” (e.g., mouse 2-OST, GENBANK® Accession No. AAC40135(SEQ ID NO:1); “3-OST-1” (e.g., human 3-OST-1, GENBANK® Accession No.NP_(—)005105 (SEQ ID NO:2); “3-OST-3” (e.g., human 3-OST-3A, GENBANK®Accession No. NP_(—)006033 (SEQ ID NO:3) and human 3-OST-3B, GENBANK®Accession No. NP_(—)006032 (SEQ ID NO:4); and “6-OST” (e.g., mouse6-OST-1, GENBANK® Accession No. NP_(—)056633 (SEQ ID NO:5), mouse6-OST-2, GENBANK® Accession No. BAA89247 (SEQ ID NO:6), and mouse6-OST-3, GENBANK® Accession No. NP_(—)056635 (SEQ ID NO:7)), which areHS 2-O-sulfotransferase, HS 3-O-sulfotransferase isoform 1, HS3-O-sulfotransferase isoform 3, and HS 6-O-sulfotransferase,respectively.

The term “OST” includes invertebrate and vertebrate homologs of theO-sulfotransferases (e.g., mammalian (such as human and mouse), insect,and avian homologs). As such, although exemplary embodiments ofparticular OSTs have been disclosed herein, the presently disclosedsubject matter is not intended to be limited to the disclosed examples,but rather “OST”, including particular OSTs (e.g., 2-OST, 3-OST-1,3-OST-3, and 6-OST), includes all comparable OSTs known to the skilledartisan.

The terms “OST gene product”, “OST protein”, and “OST polypeptide” referto peptides having amino acid sequences which are substantiallyidentical to native amino acid sequences from the organism of interestand which are biologically active in that they comprise all or a part ofthe amino acid sequence of a HS O-sulfotransferase isoform, orcross-react with antibodies raised against a HS O-sulfotransferaseisoform polypeptide, or retain all or some of the biological activity ofthe native amino acid sequence or protein. Such biological activity caninclude immunogenicity.

The terms “OST gene product”, “OST protein”, and “OST polypeptide” alsoinclude analogs of HS O-sulfotransferase molecules. By “analog” isintended that a DNA or peptide sequence can contain alterations relativeto the sequences disclosed herein, yet retain all or some of thebiological activity of those sequences. Analogs can be derived fromgenomic nucleotide sequences as are disclosed herein or from otherorganisms, or can be created synthetically. Those skilled in the artwill appreciate that other analogs, as yet undisclosed or undiscovered,can be used to design and/or construct OST analogs. There is no need fora “OST gene product”, “OST protein”, and “OST polypeptide” to compriseall or substantially all of the amino acid sequence of a native OST geneproduct. Shorter or longer sequences are anticipated to be of use in thepresently disclosed subject matter, shorter sequences are hereinreferred to as “segments.” Thus, the terms “OST gene product”, “OSTprotein”, and “OST polypeptide” also include fusion or recombinant HSO-sulfotransferase polypeptides and proteins comprising sequences of theOST protein. Methods of preparing such proteins are known in the art.For example, in some embodiments, the OST is a 2-OST or a 6-OST fusionprotein, such as a maltose-binding protein (MBP)-2-OST fusion protein ora MBP-6-OST fusion protein, as disclosed herein.

The terms “OST gene”, “OST gene sequence”, and “OST gene segment” referto any DNA sequence that is substantially identical to a polynucleotidesequence encoding a HS O-sulfotransferase isoform gene product, proteinor polypeptide as defined above, and can also comprise any combinationof associated control sequences. The terms also refer to RNA, orantisense sequences, complementary to such DNA sequences. As usedherein, the term “DNA segment” refers to a DNA molecule that has beenisolated free of total genomic DNA of a particular species. Furthermore,a DNA segment encoding a HS O-sulfotransferase polypeptide refers to aDNA segment that contains OST coding sequences, yet is isolated awayfrom, or purified free from, total genomic DNA of a source species, suchas for example Homo sapiens. Included within the term “DNA segment” areDNA segments and smaller fragments of such segments, and alsorecombinant vectors, including, for example, plasmids, cosmids, phages,viruses, and the like.

The term “substantially identical”, when used to define either a OSTgene product or amino acid sequence, or a OST gene or nucleic acidsequence, means that a particular sequence varies from the sequence of anatural OST by one or more deletions, substitutions, or additions, thenet effect of which is to retain at least some of biological activity ofthe natural gene, gene product, or sequence. Such sequences include“mutant” sequences, or sequences in which the biological activity isaltered to some degree but retains at least some of the originalbiological activity.

Alternatively, DNA analog sequences are “substantially identical” tospecific DNA sequences disclosed herein if: (a) the DNA analog sequenceis derived from coding regions of the natural OST gene; or (b) the DNAanalog sequence is capable of hybridization of DNA sequences of (a)under stringent conditions and which encode biologically active OST geneproducts; or (c) the DNA sequences are degenerate as a result ofalternative genetic code to the DNA analog sequences defined in (a)and/or (b). Substantially identical analog proteins will be greater thanabout 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thecorresponding sequence of the native protein. Sequences having lesserdegrees of identity but comparable biological activity are considered tobe equivalents. In determining nucleic acid sequences, all subjectnucleic acid sequences capable of encoding substantially similar aminoacid sequences are considered to be substantially similar to a referencenucleic acid sequence, regardless of differences in codon sequences orsubstitution of equivalent amino acids to create biologically functionalequivalents.

Sequence identity or percent similarity of a DNA or peptide sequence canbe determined, for example, by comparing sequence information using theGAP computer program, available from the University of WisconsinGeneticist Computer Group. The GAP program utilizes the alignment methodof Needleman et al. (1970) J Mol Biol 48:443, as revised by Smith et al.(1981) Adv Appl Math 2:482. Briefly, the GAP program defines similarityas the number of aligned symbols (i.e., nucleotides or amino acids) thatare similar, divided by the total number of symbols in the shorter ofthe two sequences. The preferred parameters for the GAP program are thedefault parameters, which do not impose a penalty for end gaps. SeeSchwartz et al. (1979) Nuc Acids Res 6(2):745-755; Gribskov et al.(1986) Nuc Acids Res 14(1):327-334.

In certain embodiments, the present subject matter concerns the use ofOST genes and gene products that include within their respectivesequences a sequence that is essentially that of an OST gene, or thecorresponding protein. The term “a sequence essentially as that of anOST gene”, means that the sequence is substantially identical orsubstantially similar to a portion of an OST gene and contains aminority of bases or amino acids (whether DNA or protein) which are notidentical to those of an OST protein or an OST gene, or which are not abiologically functional equivalent. The term “biologically functionalequivalent” is well understood in the art and is further defined indetail herein. Nucleotide sequences are “essentially the same” wherethey have between about 75% and about 85% or more preferably, betweenabout 86% and about 90%, or more preferably greater than 90%, or morepreferably between about 91% and about 95%, or even more preferably,between about 96% and about 99%; of nucleic acid residues which areidentical to the nucleotide sequence of a OST gene. Similarly, peptidesequences which have about 60%, 70%, 80%, or 90%, or preferably from90-95%, or more preferably greater than 96%, or more preferably 95-98%,or most preferably 96%, 97%, 98%, or 99% amino acids which are identicalor functionally equivalent or biologically functionally equivalent tothe amino acids of an OST polypeptide will be sequences which are“essentially the same”.

OST gene products and OST encoding nucleic acid sequences, which havefunctionally equivalent codons, are also covered by the presentlydisclosed subject matter. The term “functionally equivalent codon” isused herein to refer to codons that encode the same amino acid, such asthe ACG and AGU codons for serine. Applicants contemplate substitutionof functionally equivalent codons of Table 1 into sequences of OSTsdisclosed herein as equivalents.

TABLE 1 Functionally Equivalent Codons Amino Acids Codons Alanine Ala AGCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAUGlutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly GGGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUULysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU MethionineMet M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCUGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SerineSer S ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine ValV GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It will also be understood by those of skill in the art that amino acidand nucleic acid sequences can include additional residues, such asadditional N- or C-terminal amino acids or 5′ or 3′ nucleic acidsequences, and yet still be encompassed by the OSTs disclosed herein, solong as the sequence retains biological protein activity where proteinexpression is concerned. The addition of terminal sequences particularlyapplies to nucleic acid sequences which can, for example, includevarious non-coding sequences flanking either of the 5′ or 3′ portions ofthe coding region or can include various internal sequences, i.e.,introns, which are known to occur within genes.

The present subject matter also encompasses the use of nucleotidesegments that are complementary to the sequences of the present subjectmatter, in one embodiment, segments that are fully complementary, i.e.complementary for their entire length. Nucleic acid sequences that are“complementary” are those, which are base-paired according to thestandard Watson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as can be assessed by the same nucleotidecomparison set forth above, or is defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein. A particularexample of a complementary nucleic acid segment is an antisenseoligonucleotide.

One technique in the art for assessing complementary sequences and/orisolating complementary nucleotide sequences is hybridization. Nucleicacid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess ofabout 30° C., typically in excess of about 37° C., and preferably inexcess of about 45° C. Stringent salt conditions will ordinarily be lessthan about 1,000 mM, typically less than about 500 mM, and preferablyless than about 200 mM. However, the combination of parameters is muchmore important than the measure of any single parameter. See e.g.,Wethmur & Davidson (1968) J Mol Biol 31:349-370. Determining appropriatehybridization conditions to identify and/or isolate sequences containinghigh levels of homology is well known in the art. See e.g., Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

For the purposes of specifying conditions of high stringency, preferredconditions are salt concentration of about 200 mM and temperature ofabout 45° C. One example of such stringent conditions is hybridizationat 4×SSC, at 65° C., followed by a washing in 0.1×SSC at 65° C. for onehour. Another exemplary stringent hybridization scheme uses 50%formamide, 4×SSC at 42° C. Another example of “stringent conditions”refers to conditions of high stringency, for example 6×SSC, 0.2%polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1%sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA and 15% formamide at68° C. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 10×SSC (0.9 M NaCl/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1×SSC. Sequence identity can bedetermined by hybridization under stringent conditions, for example, at50° C. or higher and 0.1×SSC (9 mM NaCl/0.9 mM sodium citrate).

Nucleic acids that are substantially identical to the provided OSTs,e.g., allelic variants, genetically altered versions of the gene, etc.,bind to the disclosed OSTs under stringent hybridization conditions. Byusing probes, particularly labeled probes of DNA sequences, one canisolate homologous or related genes. The source of homologous genes canbe any species, e.g., primate species, particularly human; rodents, suchas rats and mice; canines; felines; bovines; ovines; equines; insects;yeasts; nematodes; etc.

Between mammalian species, e.g., human, mouse and rat, homologs havesubstantial sequence similarity, i.e. at least 75% sequence identitybetween nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which can be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nucleotides long, moreusually at least about 30 nucleotides long, and can extend to thecomplete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal. (1990) J Mol Biol 215:403-410. The sequences provided herein areessential for recognizing OST related and homologous proteins indatabase searches.

At a biological level, identity is just that, i.e. the same amino acidat the same relative position in a given family member of a gene family.Homology and similarity are generally viewed as broader terms. Forexample, biochemically similar amino acids, for example leucine andisoleucine or glutamate/aspartate, can be present at the sameposition—these are not identical per se, but are biochemically“similar”. As disclosed herein, these are referred to as conservativedifferences or conservative substitutions. This differs from aconservative mutation at the DNA level, which changes the nucleotidesequence without making a change in the encoded amino acid, e.g., TCC toTCA, both of which encode serine.

When percentages are referred to herein, it is meant to refer to percentidentity. The percent identities referenced herein can be generated byalignments with the program GENEWORKS™ (Oxford Molecular, Inc. ofCampbell, Calif., U.S.A.) and/or the BLAST program at the NCBI website.Another commonly used alignment program is entitled CLUSTAL W and isdescribed in Thompson et al. (1994) Nucleic Acids Res 22(22):4673-4680,among other places.

The term “gene” is used for simplicity to refer to a functional protein,polypeptide or peptide encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences and cDNAsequences.

As noted above, modifications and changes can be made in the structureof the OST proteins and peptides described herein and still constitute amolecule having like or otherwise desirable characteristics. Forexample, certain amino acids can be substituted for other amino acids ina protein structure without appreciable loss of interactive capacitywith, for example, structures in the nucleus of a cell. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence (or the nucleic acidsequence encoding it) to obtain a protein with the same, enhanced, orantagonistic properties. Such properties can be achieved by interactionwith the normal targets of the native protein, but this need not be thecase, and the biological activity of the presently disclosed subjectmatter is not limited to a particular mechanism of action. It is thuscontemplated in accordance with the present subject matter that variouschanges can be made in the sequence of the OST proteins and peptides orunderlying nucleic acid sequence without appreciable loss of theirbiological utility or activity.

Biologically functional equivalent peptides, as used herein, arepeptides in which certain, but not most or all, of the amino acids canbe substituted. Thus, applicants contemplate substitution of codons thatencode biologically equivalent amino acids as described herein into thesequences of the disclosed OSTs, but which are not set forth herein intheir entirety for convenience.

Alternatively, functionally equivalent proteins or peptides can becreated via the application of recombinant DNA technology, in whichchanges in the protein structure can be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man can be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein or to test OST mutants in order toexamine OST sulfotransferase activity, or other activity at themolecular level.

Amino acid substitutions, such as those which might be employed inmodifying the OST proteins and peptides described herein, are generallybased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. An analysis of the size, shape and type of the aminoacid side-chain substituents reveals that arginine, lysine and histidineare all positively charged residues; that alanine, glycine and serineare all of similar size; and that phenylalanine, tryptophan and tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas biologically functional equivalents. Other biologically functionallyequivalent changes will be appreciated by those of skill in the art.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al. (1982) J Mol Biol 157:105, incorporated herein byreference). It is known that certain amino acids can be substituted forother amino acids having a similar hydropathic index or score and stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 of the original value is preferred, those, whichare within ±1 of the original value, are particularly preferred, andthose within ±0.5 of the original value are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those, which are within ±1 of theoriginal value, are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges can be effected by alteration of the encoding DNA, taking intoconsideration also that the genetic code is degenerate and that two ormore codons can code for the same amino acid.

Thus, it will also be understood that the presently disclosed subjectmatter is not limited to the particular nucleic acid and amino acidsequences of the OSTs disclosed herein. Recombinant vectors and isolatedDNA segments can therefore variously include the O-sulfotransferasepolypeptide-encoding region itself, include coding regions bearingselected alterations or modifications in the basic coding region, orinclude larger polypeptides which nevertheless comprise theO-sulfotransferase polypeptide-encoding regions or can encodebiologically functional equivalent proteins or peptides which havevariant amino acid sequences. Biological activity of anO-sulfotransferase can be determined using techniques generally known inthe art, for example as disclosed herein in the Examples.

The nucleic acid segments of the present subject matter, regardless ofthe length of the coding sequence itself, can be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length can be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments can beprepared which include a short stretch complementary to a nucleic acidsequence set forth in any of the OSTs disclosed herein, such as about 10nucleotides, and which are up to 10,000 or 5,000 base pairs in length,with segments of 3,000 being preferred in certain cases. DNA segmentswith total lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100,and about 50 base pairs in length are also contemplated to be useful.

Recombinant vectors form further aspects of the present subject matter.Particularly useful vectors are those in which the coding portion of theDNA segment is positioned under the control of a promoter. The promotercan be that naturally associated with the OST gene, as can be obtainedby isolating the 5′ non-coding sequences located upstream of the codingsegment or exon, for example, using recombinant cloning and/orpolymerase chain reaction (PCR) technology and/or other methods known inthe art, in conjunction with the compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is a promoter that is not normally associated witha 3-O-sulfotransferase gene in its natural environment. Such promoterscan include promoters isolated from bacterial, viral, eukaryotic, ormammalian cells. Naturally, it will be important to employ a promoterthat effectively directs the expression of the DNA segment in the celltype chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology (See, e.g., Sambrook et al. (2001)Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). The promotersemployed can be constitutive or inducible and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides.

In some embodiments of the method disclosed herein for sulfatingpolysaccharides, the OST enzyme is immobilized on a substrate. Thisprovides an advantage in that the substrate to which the OSTs areattached can be washed after a sulfation reaction to remove allcomponents of the reaction except the bound OSTs. As such, the productsof the reaction can be more easily separated from the enzymes catalyzingthe reaction and the OSTs can be recycled and utilized again in multiplesulfation reactions. In some embodiments, the substrate is agarose. Inparticular embodiments, the agarose substrate is an agarose bead and theOSTs are linked to the beads.

II.B. Reduction of Inhibitory Effects of PAP

The presently disclosed method for sulfating polysaccharides cancomprise providing a “reaction condition” that modifies PAP to reduceinhibitory effects of PAP, such as competing with PAPS for binding withOSTs, on the polysaccharide sulfation. In some embodiments, the reactioncondition comprises a phosphatase enzyme. The phosphatase enzyme canremove a phosphate from the PAP, which reduces its binding affinity forOSTs. In some embodiments, the phosphatase is 3′-ribonucleotidephosphohydrolase.

In some embodiments, the reaction condition is a PAPS regenerationsystem, which comprises a PAPS regenerating enzyme and a sulfur donorcompound. The PAPS regenerating enzyme catalyzes regeneration of thePAPS from the PAP utilizing the sulfur donor compound as a substrate.See, e.g., U.S. Pat. No. 6,255,088; Burkart et al., (2000) J. Org. Chem.65, 5565-5574, which is herein incorporated by reference in itsentirety. Thus, the PAPS regeneration system provides the dualadvantages of reducing the inhibitory effects of PAP accumulation onsulfotransferase activity while also constantly “recharging” thereaction mixture with the primary sulfur donor molecule, PAPS.

Thus, an aspect of the presently disclosed subject matter is directed toa sulfur donor compound (e.g., PAPS) regeneration process coupled withsulfation of a polysaccharide substrate. In particular, the process canbe of a type wherein the sulfation of a polysaccharide substrate iscatalyzed by a sulfotransferase, such as one or more OSTs, with aconversion of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to adenosine3′,5′-diphosphate (PAP). The sulfation process can be coupled with anenzymatic regeneration of the 3′-phosphoadenosine-5′-phosphosulfate fromthe adenosine 3′,5′-diphosphate. The enzymatic regeneration can employan arylsulfotransferase as the catalyst and an aryl sulfate as asubstrate. As previously disclosed, preferred carbohydrate substratescan include GAGs, such as for example heparan sulfates, includingheparin.

In some embodiments, the arylsulfotransferase is a recombinant arylsulfotransferase IV (AST-IV; e.g., rat AST-IV (SEQ ID NO:8)). Thisenzyme, when coupled to a sulfotransferase of choice, transfers sulfatefrom an aryl sulfate (e.g., p-nitrophenyl sulfate (PNPS) to PAP. Thissystem averts product inhibition by PAP while regenerating PAPS in situand can be monitored quantitatively by measurement of the absorbance ofreleased p-nitrophenol at 400 nm.

The enzyme AST-IV exists in two oxidative forms (Marshall et al., (1997)J. Biol. Chem. 272, 9153-9160; Marshall et al., (1998) Chem.-Biol.Interact. 109, 107-116; Yang et al., (1998) Chem.-Biol. Interact. 109,129-135; Yang et al. (1996) Protein Expression Purif. 8, 423-429; Guo etal. (1994) Chem.-Biol. Interact. 92, 25-31; Chen et al. (1992) ProteinExpression Purif. 3, 421-6; Lin et al. (1998) Anal. Biochem. 264,111-117; and Yang et al., (1997) Protein Eng. 10, 70). These twooxidative forms can be easily resolved (Yang et al. (1996) ProteinExpression Purif. 8, 423-429), and the resolved physiologically relevantform has been utilized to assay picomole quantities of PAPS and PAP (Linet al. (1998) Anal. Biochem. 264, 111-117). As the bacterial expressionof rat AST-IV has been demonstrated (Chen et al., (1992) ProteinExpression Purif. 3, 421-6; and Ozawa et al., (1990) Nucleic Acids Res.18, 4001 z.), AST IV can be cloned from a rat liver cDNA library,overexpressed in a recombinant bacterial system (e.g., E. coli) andisolated (See, e.g., U.S. Pat. No. 6,255,088, herein incorporated byreference in its entirety).

Coupling the sulfotransferase catalyzed sulfation reaction with a PAPSregeneration system can provide a further advantage of generating PAPSutilized in the reaction directly from PAP. That is, the reactionmixture can be formulated to combine PAP with a PAPS regenerating enzymeprior to or simultaneously with addition of a sulfotransferase to thereaction mixture. The PAPS regenerating enzyme can then generate PAPSfrom the PAP for use by the sulfotransferase, thereby alleviating theneed of supplying any of the more expensive and unstable PAPS to thereaction mixture. As such, in some embodiments of the presentlydisclosed subject matter a method of sulfating a polysaccharide isprovided comprising providing a reaction mixture comprising thereinadenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and asulfur donor compound (other than PAPS) and incubating the reactionmixture for a time period sufficient to catalyze the production of3′-phosphoadenosine 5′-phosphosulfate (PAPS) from the PAP by the PAPSregenerating enzyme utilizing the sulfur donor compound as a substrate.The method further comprises incubating a polysaccharide substrate andat least one O-sulfotransferase (OST) enzyme with the reaction mixture,wherein production of a sulfated polysaccharide from the polysaccharidesubstrate is catalyzed by the OST enzyme with a conversion of the PAPSto PAP and wherein the PAPS regenerating enzyme then catalyzesregeneration of the PAPS from the PAP, again utilizing the sulfur donorcompound as a substrate.

III. Kits

The presently disclosed subject matter further provides kits forsulfating polysaccharides. In some embodiments, the kit comprises atleast one sulfotransferase enzyme (e.g., at least one OST); and areagent which modifies adenosine 3′,5′-diphosphate (PAP) to reduce aninhibitory effect of PAP on the polysaccharide sulfation. In someembodiments of the kit the at least one sulfotransferase enzyme iscontained within a first container and the reagent is contained within asecond container. The kit can further comprise instructions forsulfating a polysaccharide.

In some embodiments of the kit, the at least one sulfotransferase enzymeis an OST enzyme selected from the group consisting of 2-OST, 3-OST-1,3-OST-3, 6-OST, and combinations thereof. In some embodiments, the OSTenzyme is a recombinant OST enzyme, such as for example a recombinantOST enzyme produced in a bacterial expression system. In someembodiments, the OST enzyme is a fusion protein, such as for example aMBP-2-OST fusion protein or a MBP-6-OST fusion protein. Further, in someembodiments, the OST enzyme is immobilized to a substrate, such as forexample an agarose bead.

In some embodiments of the kit, the reagent comprises a PAPSregeneration system comprising a PAPS regenerating enzyme (e.g., AST-IV)and a sulfur donor compound (e.g., PNPS). In other embodiments of thekit, the reagent comprises a phosphatase enzyme (e.g., 3′-ribonucleotidephosphohydrolase).

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlyclaimed subject matter.

Materials and Methods for Examples 1-3

Preparation of Chemically Desulfated N-Sulfated Heparin (CDSNS Heparin).Treatment of heparin with a dimethyl sulfoxide/methanol mixture (9:1,v/v) at 80° C. for 24 h resulted in nearly complete N,O-desulfation with<5% of the solvolytically resistant O-sulfo groups remaining. ChemicalN-sulfation with sulfur trioxide-pyridine afforded CDSNS heparin, whichwas characterized by disaccharide analysis and ¹H and two-dimensionalCOSY NMR.

Expression and Purification of 2-OST and 6-OST. The catalytic domains of2-OST of Chinese hamster ovary (Arg⁵¹-Asn³⁵⁶) and mouse 6-OST-1(His⁵³-Trp⁴⁰¹) were cloned into a pMAL-c2X vector (New England Biolabs,Beverly, Mass., U.S.A.) using the BamHI and HindIII sites to generatemaltose-binding protein (MBP)-2-OST and MBP-6-OST fusion proteins. Thefull-length cDNAs of 2-OST and 6-OST-1 were gifts from Dr. Rosenberg(Massachusetts Institute of Technology, Cambridge, Mass., U.S.A.) andDr. Kimata (Aichi University, Japan), respectively. Expression of 2-OSTand 6-OST was achieved in Rosetta-gami B (DE3) cells (Novagen, a brandof EMD Biosciences, San Diego, Calif., U.S.A.) using a standardprocedure. Briefly, cells containing the plasmid expressing 2-OST or6-OST were grown in Luria broth (LB) medium supplemented with 2 mg/mlglucose, 15 μg/ml tetracycline, 15 μg/ml kenamycin, 35 μg/mlchloramphenicol, and 50 μg/ml carbenicillin at 37° C. When the A₆₀₀reached 0.6-0.8, Isopropyl-β-D-thiogalactopyranoside (1 mM) was added,and the cells were shaken overnight at 22° C. The bacteria wereharvested, and the proteins were purified by following a protocol fromthe manufacturer (New England Biolabs). The purified proteins migratedat 75 kDa on 12% SDS-PAGE with the purity greater than 80%.

Preparation of 3-OST-1 bacterial expression plasmid (b3-OST-1-pET28).The cDNA fragment encoding the catalytic domain of 3-OST-1 (G48-H311)was amplified from m3-OST-1-pcDNA3 with a 5′ overhang containing an NdeIsite and a 3′ overhang containing an EcoRI site. This construct wasinserted into the pET28a vector (Novagen) using the NdeI and EcoRIrestriction sites to produce a (His)₆-tagged protein. The resultantplasmid (b3-OST-1-pET28) was sequenced to confirm the reading frame andthe lack of mutations within the coding region (University of NorthCarolina, DNA sequencing core facility). The plasmid, b3-OST-1-pET28,was transformed into BL21(DE3)RIL cells (Stratagene, La Jolla Calif.,U.S.A) for the expression of 3-OST-1.

Protein expression and purification of 3-OST-1. Cells containing theb3-OST-1-pET28 were grown in twelve 2.8 L Fernbach flasks containing 1 Lof LB media with 50 μg/mL of kanamycin at 37° C. When the OD₆₀₀ reached0.6 to 0.8, the temperature was lowered to 22° C. for 15 min.Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a finalconcentration of 200 μM, and the cells were allowed to shake overnight.Cells were pelleted and resuspended in 120 mL of sonication buffer, 25mM Tris pH 7.5, 500 mM NaCl, and 10 mM imidizole. Cells were disruptedby sonication then spun down. The supernatant was applied to NTA-agaroseresin (Qiagen, Valencia, Calif., U.S.A.) in batch and washed withsonication buffer. The resin was loaded onto a column and the proteinwas eluted with an imidizole gradient from 10 mM to 250 mM.

Preparation of 3-OST-3 expression plasmid. The cDNA fragment encodingthe catalytic domain of 3-OST-3 (G139-G406) was amplified from plasmidh3-OST-3A-pcDNA3 (Liu, J. et al (1999) J. Biol. Chem. 274, 5185-5192)with a 5′ overhang containing an NdeI site and a 3′ overhang containingan EcoRI site. This construct was inserted into the pET28a vector(Novagen) using the NdeI and EcoRI restriction sites to produce a(His)₆-tagged protein. The resultant plasmid was sequenced to confirmthe reading frame and the lack of mutations within the coding region(University of North Carolina, DNA sequencing core facility). Theplasmid was transformed into BL21 (DE3)RIL cells (Stratagene) for theexpression of 3-OST-3.

Protein expression and purification of 3-OST-3. Cells containing the3-OST-3 were grown in twelve 2.8 L Fernbach flasks containing 1 L of LBmedia with 50 μg/mL of kanamycin at 37° C. When the OD₆₀₀ reached 0.6 to0.8, the temperature was lowered to 22° C. for 15 min.Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a finalconcentration of 200 μM, and the cells were allowed to shake overnight.Cells were pelleted and resuspended in 120 mL of sonication buffer, 25mM Tris pH 7.5, 500 mM NaCl, and 10 mM imidizole. Cells were disruptedby sonication then spun down. The supernatant was applied to NTA-agaroseresin (Qiagen) in batch and washed with sonication buffer. The resin wasloaded onto a column and the protein was eluted with an imidizolegradient from 10 mM to 250 mM.

Preparation of Immobilized Hs Sulfotransferases. Dialyzedsulfotransferases (3 ml, 4 mg/ml) in phosphate-buffered saline buffer (3mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, 138 mM NaCl, pH 7.2) were mixedwith 1 ml of AMINOLINK® plus beads (Pierce, Rockford, Ill., U.S.A.)following a protocol from the manufacturer. Immobilized enzyme waswashed with 50 mM MES, 1% Triton X-100, 1 mM MgCl₂, and 1 mM MnCl₂, pH7.0, and stored at 4° C.

Measurement of Enzymatic Activities of Immobilized Proteins. Todetermine the activity of 3-OST-1 and 3-OST-3, HS from bovine kidney(ICN Biomedicals, Aurora, Ohio, U.S.A.) was used as a substrate; todetermine the activity of 6-OST and 2-OST, CDSNS heparin was used as asubstrate. Immobilized proteins (100 μl, ˜300 μg of immobilized enzyme)with 100 μg of substrate (HS for 3-OST and CDSNS heparin for 2-OST or6-OST) and 200 μM [³⁵S]PAPS (1000 cpm/pmol) in 1 ml of 50 mM MES, pH7.0, 1% Triton X-100, 1 mM MgCl₂, and 1 mM MnCl₂. After rotating at roomtemperature for 1 h, the supernatant was collected, and the beads werewashed with 3×200 μl of 1 M NaCl in 25 mM MOPS (pH 7.0). The supernatantand washes were combined, diluted with 2 ml of water, and subjected toDEAE chromatography to determine the amount of [³⁵S]HS product used todetermine the activities of the various HS O-STs.

PAPS Regeneration System. The reactions involved in the PAPSregeneration system are shown in FIG. 1B. N-terminal His₆-tagged AST-IVwas expressed in E. coli and purified as described by Burkat andcolleagues (Burkart et al. (2000) J. Org. Chem. 65, 5565-5574) at ayield of ˜50 mg/liter of bacterial culture. The full-length cDNA of ratAST-IV was a generous gift of Dr. Michael Duffel (University of Iowa)(Sheng et al. (2004) Drug Metabol. Dispos. 32, 559-565).

Modification of Polysaccharides. All enzymatic modifications, includingthose catalyzed by 2-OST, 6-OST, 3-OST-1, and 3-OST-3 followed the sameprotocol. Briefly, 20 mg of purified AST-IV was incubated with 40 μM PAPand 1 mM PNPS in 20 ml of 50 mM MES, pH 7.0, 1% Triton X-100, 1 mMMgCl₂, and 1 mM MnCl₂ at 25° C. for 15 min. The reaction mixture wasmixed with 4 ml of immobilized sulfotransferase, 2 mg of apolysaccharide substrate was added, and the mixture was rotated at 25°C. for 24 h. The supernatant was recovered, and the polysaccharide thatbound to the beads was eluted by washing three times with 8 ml of 1 MNaCl in 25 mM MOPS. Both the supernatant and wash were combined and theproduct was precipitated by adding ethanol (3 volumes). After incubatingovernight at 4° C., polysaccharide was recovered by centrifugation.

Estimation of the Modification Level. The completion of 2-OSTmodification was monitored by incubating 10 μg of polysaccharide (Liuand Thorp (2002) Med. Res. Rev. 22, 1-25) with 20 μg of soluble 2-OST inthe presence of 100 μM of [³⁵S]PAPS at 37° C. for 30 min. In a controlexperiment, 10 μg of compound 1 (FIG. 1A) replaced the previouslymodified polysaccharide in the otherwise identical reaction mixture. Bycomparing the amount of ³⁵S incorporation, the extent of the original2-O-sulfation reaction could be estimated. For monitoring the completionof 6-OST, compound 1 or 2 (FIG. 1A) was used as a substrate; for3-OST-1, compound 4 was used (FIG. 1A).

Using [³⁵S]PAPS as a Sulfate Donor. Antithrombin (AT)-binding andgD-binding experiments utilized ³⁵S-labeled polysaccharides preparedusing [³⁵S]PAPS. In a typical reaction, 2 mg of HS substrate wasincubated with 4 ml of beads with immobilized sulfotransferase (˜12 mgof immobilized enzyme) at 25° C. in 20 ml of 50 mM MES, pH 7.0, 1%Triton X-100, 1 mM MgCl₂, and 1 mM MnCl₂, 200 μM [³⁵S]PAPS (1000cpm/pmol) for 1 h. The resultant polysaccharide was recovered using DEAEchromatography.

Disaccharide Analysis. Synthesized polysaccharides (100 μg) weredegraded by a mixture of heparin lyases as previously described (Moon etal. (2204) J. Biol. Chem. 279, 45185-45193) and desalted on BIOGEL® P-2column (0.5×200 cm; Bio-Rad Labs) in 0.1 M NH₄HCO₃. Disaccharides wereanalyzed by a C₁₈ reversed phase column (0.45×25 cm; Vydac, Columbia,Md., U.S.A.) with UV 232 detection and identified by coelution withappropriate standards (Chen et al. (2003) Glycobiology 13, 785-794). Theoverall recovery yield of the disaccharide analysis was estimated byusing 2-O-[³⁵S]heparin (100,000 cpm/70 ng; compound 2) as an internalcontrol.

NMR Analysis. Polysaccharide sample (1-2 mg) was dissolved in 0.5 ml of²H₂O (99.9%), freeze dried to remove exchangeable protons, redissolvedin 75 μl of ²H₂O (100.00%), and transferred to NMR microtubes (Shigemi,Inc., Allison Park, Pa., U.S.A.). NMR spectra were referenced relativeto the HO²H at 4.80 ppm, and in COSY water was suppressed bypresaturation of the HO²H resonance.

Determination of the Binding to AT and FGF2 Using Surface PlasmonResonance Spectroscopy (SPR). Heparin and the synthesizedpolysaccharides were biotinylated as described (Hernaiz et al. (2000)Biochem. Biophys. Res. Commun. 276, 292-297). A solution of thebiotinylated polysaccharides (0.1 mg/ml) in HEPES (10 mM), 0.15 M NaCl,3 mM EDTA, and 0.005% surfactant P20 (HBS-EP) was flowed over the cellsof the streptavidin chip at 5 μl/min. To determine the K_(D) values,different concentrations (25-1600 nM in HBS-EP buffer) of FGF2 and ATwere individually injected at 30 μl/min (running buffer: HBS-EP).Kinetic injection was used, leading the protein to flow for 180 seconds(s) and dissociated for the next 180 s, and the surface was regeneratedby a 60 s injection of 30 μl of 2 M NaCl. Response units were monitoredas a function of time to afford sensorgrams. The SPR curves for FGF2 fitwell at individual concentrations, but the global fit suggestedsignificant binding heterogeneity. Thus, the equilibrium response unitRU(eq) values from the sensorgrams of FGF2 binding topolysaccharide-containing surfaces were used to construct Scatchardplots, RU(eq)/C versus RU(eq), where C is the free proteinconcentration, resulting in linear, first degree polynomial functionsconfirming the one-to-one binding of FGF-polysaccharide and to estimatebinding affinity. A two-state reaction model was applied to theAT-polysaccharide interactions measured by SPR using curve fitting toestimate the association and dissociation rate constants and affinityconstant.

FGF2/FGFR1C-mediated Proliferation Assay. The BaF3 cells ectopicallyexpressing FGFR1c have been previously described (Ornitz et al. (1996)J. Biol. Chem. 271, 15292-15297). The BaF3-FGFR1c cells were maintainedin RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo., U.S.A.)supplemented with 10% fetal bovine serum, 0.5 ng/ml interleukin (IL)-3(PeproTech Inc., Rocky Hill, N.J., U.S.A.), 2 mM L-glutamine, penicillin(50 IU/ml) and streptomycin (50 μg/ml), and 50 μM β-mercaptoethanol. Formitogenic assays, BaF3 FGFR1c cells were washed three times with RPMI1640 medium to remove IL-3 and resuspended in the growth medium lackingIL-3. About 30,000 cells were plated per well in a 96-well plate inmedium containing 1 μg/ml heparin, compound 1, 2, 3, 4a, or 4b, and 2 nMFGF-2 (PeproTech) in a total volume of 200 μl. The cells were thenincubated at 37° C. for 40 h. To each well, an additional 50 μl ofgrowth medium containing 1 μCi of [³H]thymidine was added. Cells wereharvested after 4-5 h by filtration through glass fiber paper. Theincorporation of [³H]thymidine into the DNA was determined byscintillation counting.

Determination of the Binding of Compounds 5-8 to AT Approximately 1×10⁵cpm of ³⁵S-labeled compound was incubated with 5 μg of human AT (CutterBiological, Clayton, N.C., U.S.A.) in 50 μl of binding buffer containing10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Mn²⁺, 1 mM Mg²⁺, 1 mM Ca²⁺, 10μM dextran sulfate, 0.0004% Triton X-100, and 0.02% sodium azide for 30min at room temperature. Concanavalin A-Sepharose (Sigma; 50 μl of 1:1slurry) was then added, and the reaction was shaken at room temperaturefor 1 h. The beads were then washed by 3×1 ml of binding buffer, and thebound polysaccharide was eluted with 1 M NaCl.

Inhibition Effect of the Polysaccharides on the Activities of Factor Xaand Thrombin. Assays were based on two previous methods (Zhang et al.(2001) J. Biol. Chem. 276, 42311-42321; Duncan et al. (2004) Biochim.Biophys. Acta 1671, 34-43). Briefly, factor Xa (Enzyme ResearchLaboratories, South Bend, Ind., U.S.A.) and thrombin (Sigma) werediluted to 20 and 8 units/ml with phosphate-buffered saline containing 1mg/ml bovine serum albumin, respectively. AT was diluted withphosphate-buffered saline containing 1 mg/ml bovine serum albumin togive a stock solution at the concentration of 27 μM. The chromogenicsubstrates, S-2765 (for factor Xa assay) and S-2238 (for thrombin assay)were from Diapharma (West Chester, Ohio, U.S.A.) and made up at 1 mMwith 1 mg/ml POLYBRENE® (Sigma) in water. The synthesized polysaccharide(compounds 5, 7, and 8; FIG. 1A) or heparin was dissolved in a buffercontaining 50 mM Tris-HCl, pH 8.4, 7.5 mM Na₂EDTA, 175 mM NaCl atvarious concentrations (1-10,000 ng/ml). The reaction mixture, whichcomprised 25 μl of AT stock solution and 25 μl of the solutioncontaining polysaccharide, was incubated at 37° C. for 2 min. Factor Xa(25 μl) or thrombin (25 μl) was added. After incubating 37° C. for 4min, 25 μl of S-2765 or S-2238 was added. The absorbance of the reactionmixture was measured at 405 nm continuously for 10 min. The absorbancevalues were plotted against the reaction time. The initial reactionrates as a function of concentration were used to calculate the IC₅₀values. The concentrations of the synthesized polysaccharides weredetermined using Alcian blue as described by Bjornsson (Bjornsson (1993)Anal. Biochem. 210, 282-291) and quantitative disaccharide analysis asdescribed above.

The Binding to Herpes Simplex Virus gD. The assay for determiningbinding of ³⁵S-labeled polysaccharides (compounds 5 and 6; FIG. 1A) togD was carried out by an immunoprecipitation procedure using anti-gDmonoclonal antibody (DL6) (Shukla et al. (1999) Cell 99, 13-22).

Example 1 Development of an Efficient Enzymatic Synthesis of SulfatedPolysaccharides

Expression of HS Sulfotransferases in E. coli. The presently disclosedsubject matter provides methods for synthesizing biologically active HS,such as for example FGF-binding HS (e.g., compound 4, including 4a and4b), AT-binding HS (e.g., compound 5), and herpes simplex virusgD-binding HS (e.g., compound 6). See FIG. 1A. Four enzymes, including2-OST, 6-OST, 3-OST-1, and 3-OST-3, were utilized for the syntheses ofthese particular targets. Bacterial expressed 3-OST-1 and 3-OST-3 canexhibit substrate specificity and specific enzymatic activity comparablewith those of their counterparts expressed in insect cells (Moon et al.(2004) J. Biol. Chem. 279, 45185-45193; Edavettal et al. (2004) J. Biol.Chem. 279, 25789-25797). Expression of the catalytic domains of 2-OSTand 6-OST was also achieved in relatively high yield by preparing afusion protein with MBP and 2-OST or 6-OST in Rosetta-gami B cells.Because 2-OST and 6-OST fusion proteins were enzymatically active andhighly soluble, the MBP domain was retained.

Immobilized Enzymes Are Reusable. 2-OST, 6-OST, 3-OST-1, and 3-OST-3were immobilized on agarose to be reusable and to enhance the thermalstability. Immobilized enzymes were incubated with polysaccharidesubstrate and PAPS for 1 h at room temperature. The sulfatedpolysaccharide product was separated from the immobilized enzyme bywashing the beads with 1 M NaCl followed by centrifugation, making theimmobilized enzymes ready for the next synthetic cycle. The cycle wasrepeated 10 times, after which each of the immobilized enzymes wereassayed and showed >80% of their catalytic activity (FIG. 2). It wasalso determined that the immobilized enzymes also maintained >65% oftheir catalytic activity after 2 months of storage at 8° C.

Introduction of PAPS Regeneration System. PAP inhibitssulfotransferase-catalyzed reactions. A PAPS regeneration system can beused to convert PAP to PAPS by relying on AST-IV to catalyze thetransfer of the sulfo group from PNPS to PAP, as illustrated in FIG. 1B.The presently disclosed subject matter provides for the use of the PAPSregeneration system coupled with O-sulfotransferases. As disclosedherein, the PAPS regeneration system performed very well with 2-OST,6-OST, and 3-OST-1. Complete modification of the substrate could bedemonstrated by the low susceptibility of polysaccharide product toundergo additional sulfation using [³⁵S]PAPS with soluble enzymes, asdisclosed herein above. Under the standard conditions, 2-OST, 6-OST, and3-OST-1 afforded 98, 97, and 98% complete modification, respectively,using the PAPS regeneration system. These results demonstrate that thePAPS regeneration system functioned properly, providing sufficient PAPSfor the complete sulfotransferase-catalyzed modification ofpolysaccharide substrates. This conclusion was further supported bycharacterizing the structures of the newly synthesized polysaccharideproducts 2, 3, 4a, 4b, and 5 (described herein below; see also FIG. 1A).In a parallel experiment, [³⁵S]PAPS was used as the sulfo donor tosulfate the polysaccharides in order to estimate the number of sulfogroups that were incorporated into the product. Six 2-O-sulfo and6-O-sulfo groups and three 3-O-sulfo groups were estimated to betransferred to one polysaccharide molecule, respectively, assuming thatthe length of the polysaccharide is 25 disaccharide units. Approximately1.5 mg of anticoagulant HS (5) was synthesized from 10 mg of CDSNSheparin (1) using immobilized sulfotransferases and the PAPSregeneration system.

Example 2 Structural Characterization of Synthesized Polysaccharides

Disaccharide Analysis of the Polysaccharides. Synthesized polysaccharideintermediates were digested with a mixture of heparin lyases, and theresulting disaccharides were analyzed using RPIP-HPLC (FIG. 3). Asexpected, analysis of compound 1 showed a disaccharide component of thestructure of ΔUA-GlcNS (disaccharide 2), residual unsulfateddisaccharide, ΔUA-GlcNAc (disaccharide 1), and small amounts of sulfateddisaccharides (disaccharides 3-5) due to incomplete chemical desulfation(FIG. 3A and Table 2). The modification by 2-OST elevated the level ofΔUA2S-GlcNS (disaccharide 4) by about 5-fold (FIG. 3B and Table 2),confirming the structure of compound 2. Analysis of compound 3 affordeda 7-fold increase in the level of the disaccharide ΔUA-GlcNS6S(disaccharide 3) compared with compound 1, consistent with6-OST-catalyzed modification (FIG. 3C and Table 2). The level oftrisulfodisaccharide, ΔUA2S-GlcNS6S (disaccharide 5), in compounds 4aand 4b was increased by about 10-fold compared with that of compound 1(FIGS. 3D and 3E, and Table 2). The results from the disaccharideanalysis clearly establish that the expected enzymatic modificationstook place at each step. It is interesting to note that 6-O-sulfationoccurs at N-sulfoglucosamine, consistent with the substrate specificityof 6-OST in vitro (Smeds et al. (2003) Biochem. J. 372, 371-380). The2-O-sulfation predominantly occurs at the uronic acid with anN-sulfoglucosamine residue at the reducing end. It is also noted thatsubstantial amounts of ΔUA-GlcNS (disaccharide 2) remain in compounds 4aand 4b. This observation is not unexpected, since the HS from varioustissues also afford a ΔUA-GlcNS disaccharide unit, suggesting that thestructures of compounds 4a and 4b are similar to HS from natural sources(Ledin et al. (2004) J. Biol. Chem. 279, 42732-42741).

TABLE 2 Summary of the disaccharide compositions of the syntheticpolysaccharides ΔUA-GlcNAc ΔUA-GlcNS ΔUA2S-GlcNS ΔUA-GlcNS6SΔUA2S-GlcNS6S Total recovery Compound nmol^(a) % nmol % nmol % nmol %nmol % %^(b) 1 12.5 (16.1%) 50.0 (64.4%) 6.3 (8.1%) 6.3 (8.1%) 2.5(3.2%) 54.0 2 12.5 (16.6%) 27.5 (36.6%) 30.0 (40.0%) 2.5 (3.3%) 2.5(3.3%) 49.2 3 10.0 (14.0%) 27.5 (38.5%) 5.0 (7.0%) 25.0 (35.0%) 3.8(5.3%) 51.0 4a 12.5 (18.8%) 17.5 (26.2%)  7.5 (11.2%) 10.0 (15.0%) 20.0(30.0%) 50.0 4b 17.5 (21.7%) 15.0 (18.6%) 8.0 (9.9%) 15.0 (18.6%) 25.0(31.0%) 48.0 ^(a)The amount of each disaccharide was estimated bydetermining its peak area with a standard curve generated with a knownamount of the disaccharide standard. ^(b)A recovery yield was calculatedby using 2-O—[³⁵S]sulfoheparin as an internal standard.

Each synthesized polysaccharide (100 μg) was digested with a mixture ofheparin lyases. The resultant disaccharides were purified by a BioGelP-2 column and resolved by RPIP-HPLC.

NMR Analysis of the Polysaccharides. The ¹H NMR analysis was conductedon the synthesized polysaccharides to allow assessment of theirstructures at the polymer levels as well as to estimate the composition.The assignment of each non-exchangeable proton was made using COSY, andtheir chemical shifts are reported in Table 3.

TABLE 3 ¹H chemical shift data (in ppm) for the synthesizedpolysaccharides GlcN GlcN GlcN GlcN GlcN GlcN IdoUA IdoUA IdoUA IdoUAIdoUA H1 H2 H3 H4 H5 H6a/b H1 H2 H3 H4 H5 Heparin 5.41 3.26 3.69 3.794.04 4.29 5.22 4.35 4.22 4.12 4.82 4.42 compound 1 5.40 3.23 3.67 3.723.79 3.84 4.87 3.70 3.93 4.17 4.72 compound 2 5.33 3.24 3.70 3.743.75-3.84 3.85 5.23 4.34 4.23 4.08 4.87 compound 3 5.38 3.24 3.63 3.784.13 4.30 4.95 3.72 4.10 4.05 4.74 compound 4a 5.38 3.25 3.70 3.83 4.194.25 5.23 4.33 4.21 4.13 4.90 4.33 compound 4b 5.37 3.25 3.67 3.83 4.184.26 5.22 4.35 4.21 4.15 4.90 4.33 compound 5 5.36 3.22 4.10 3.71 4.174.32 5.23 4.33 4.22 4.08 4.94 4.38

The integration of the N-acetyl signal in the ¹H NMR of heparin wascompared with the integration of the GlcNS-H1 signal, allowingestimation of the level of modifications. The ratio of these integralsshowed that ˜15% of the glucosamine residues contained N-acetyl groups.Using this information, the incorporation of 2-O-, 3-O-, and 6-O-sulfogroups was examined. Comparison of the N-acetyl peak with IdoUA2S-H2 incompound 2 demonstrates incorporation of a 2-O-sulfo group into 35% ofthe IdoUA residues. Based on this incorporation into compound 2, theintegral of GlcNS-H6a and IdoUA2S-H2 (overlapping signal) was comparedwith the integral of the N-acetyl methyl group in compound 4a toestimate the level of incorporation of the 6-O-sulfo group. Integrationshowed that the 6-O-sulfo group was incorporated into 25% of the GlcNSresidues. The reduced level of incorporation is not surprising, sinceonly the 6-OST-1, and not 6-OST-2 and -3, was used in this synthesis. Incompound 5, the incorporation of the 3-O-sulfo group was calculated bycomparing the integral of the N-acetyl methyl group with GlcN-H3 andIdoUA-H4 (overlapping signal). Based on these calculations, the3-O-sulfo group was incorporated into 32% of the GlcNS6S residues.According to ¹H NMR and COSY experiments (Table 3), the structures ofthe compounds 4a, 4b, and 5 were all found to be similar to heparin andcontained no unusual signals. Compound 5 showed a slightly greaterheterogeneity, as evidenced by additional minor signals corresponding toadditional 3-O-sulfo group-containing sequences in the ¹H NMR, whencompared with heparin. This is not unexpected, since heparin contains alower level of 3-O-sulfo groups/chain than the content of 3-O-sulfogroups/chain observed in compound 5 as determined by ³⁵S incorporation.

The composition estimated by NMR is consistent with the results ofdisaccharide analysis. NMR analysis suggests that about 15% of theglucosamine unit is N-acetylated, which is similar to the results ofdisaccharide analysis for ΔUA-GlcNAc (14-22%; Table 2). NMR analysisdemonstrates that compound 2 comprises 35% of IdoUA2S-GlcNS, andcompound 4a comprises 25% of IdoUA2S-GlcNS6S, and disaccharide analysisdemonstrates that compounds 2 and 4a comprise 40% of ΔUA2S-GlcNS and 30%of ΔUA2S-GlcNS6S, respectively. Because of the signal overlap, thedegree of 6-O-sulfation in compounds 3 and 4b using ¹H NMR was notcalculated.

Example 3 Determination of the Biological Activities of the SynthesizedPolysaccharides

The Binding of the Polysaccharides to AT and FGF2. Characterization ofthe affinities of AT to heparin and enzymatically modified heparinderivatives were performed by SPR. A two-state reaction model wasapplied to the SPR study of AT and polysaccharide interactions usingBIAevaluation™ Software (Biacore Life Sciences, Uppsala, Sweden) forcurve fitting analysis. None of the derivatives with the exception ofthe 2,6,3-O-sulfopolysaccharide (5) and heparin had high affinity to AT.The binding constant (K_(D)) for the binding of compound 5 to AT wasdetermined to be 170 nM, which is very similar to that of heparin (75nM). The binding affinity of FGF2 to the synthesized polysaccharides wasalso estimated. Compound 1 showed no interaction with FGF2, whereascompounds 4a and 4b showed the identical binding affinity to FGF2 at aK_(d) of 35 nM, which is similar to heparin (22 nM) and is consistentwith the value reported in the literature (Ibrahimi et al. (2004)Biochemistry 43, 4724-4730).

Synthetic Polysaccharides Promote Cell Proliferation. The BaF3 FGFR1ccells normally depend on IL-3 for growth. In the absence of IL-3, thecell proliferation depends on the addition of both FGF and heparin or HS(Ornitz et al. (1996) J. Biol. Chem. 271, 15292-15297). The activity ofcompounds 1, 2, 3, 4a, and 4b and heparin in promoting cell mitogenesiswas measured using the FGF-2/FGFR1c system in BaF3 cells as describedherein above. The cells receiving compounds 4a and 4b (at 1 μg/ml)showed an increase in [³H]thymidine incorporation, which was about 60and 40% of that of heparin, respectively, suggesting that thecombinations of 2-O- and 6-O-sulfations confer the activity in promotingcell proliferation (FIG. 4A). The activity of 4a and 4b was alsocompared with that of heparin at different concentrations (FIG. 4B). Itwas found that compound 3 had moderate activity in promoting cellproliferation, whereas compounds 1 and 2 did not exhibit activity. Theresults are consistent with previously reported data on thecontributions of the sulfo groups of HS to the mitogenic activity(Guimond and Turnbull (1999) Curr. Biol. 9, 1343-1346; Pye et al. (1998)J. Biol. Chem. 273, 22936-22942). It is noted that 4a appears to havestronger activity in promoting cell proliferation than 4b, suggestingthat different sequences are generated by the different order of 2-O-and 6-O-sulfation as suggested by Jacobsson and Lindahl (Jacobsson andLindahl (1980) J. Biol. Chem. 255, 5094-5100). However, such sequencedifference could not be detected by the disaccharide or NMR analysis.

The Anticoagulant Activity of the Synthesized Polysaccharides. Heparinachieves its anticoagulant activity by forming a 1:1 complex with AT,which inhibits the activities of factor Xa and thrombin (Rosenberg etal. (1997) J. Clin. Invest. 99, 2062-2070). Because it is known that theintroduction of the 3-O-sulfo group by 3-OST-1 is essential for thesynthesis of anticoagulant HS, different types of 3-O-sulfogroup-containing polysaccharides, compounds 5, 7, and 8, were preparedand their activities in inhibiting factor Xa and thrombin tested (Table4). As expected, heparin is a potent activator for AT-mediatedinhibition of factor Xa and thrombin, whereas ARIXTRA® (GlaxoSmithKline)specifically activates the AT-mediated inhibition of factor Xa (Petitouand van Boeckel (2004) Angew. Chem. Int. Ed. 43, 3118-3133). Compound 5has very similar potency to heparin, inhibiting the activities of bothfactor Xa and thrombin, suggesting that the presently disclosedenzyme-based approach is indeed capable of synthesizing theanticoagulant polysaccharide. It has been reported that the presence of2-O-sulfo groups is not essential for HS binding to AT and its resultinganticoagulant activity (Zhang et al. (2001) J. Biol. Chem. 276,28806-28813). Indeed, polysaccharide intermediate compound 8 lacks2-O-sulfo groups but still exhibits anticoagulant activity, consistentwith this previous report (Zhang et al. (2001) J. Biol. Chem. 276,28806-28813). In contrast, compound 7 lacks 6-O-sulfo groups and, thus,has no anticoagulant activity, since 6-O-sulfo groups are critical in ATbinding (Atha et al. (1985) Biochemistry 24, 6723-6729). Another3-O-sulfated polysaccharide (compound 6) was also prepared to test forits anti-Xa and antithrombin activities. It is important to note thatboth compounds 5 and 6 carry a 3-O-sulfoglucosamine unit, although it islocated in different saccharide sequences (FIG. 1A). It is known that3-OST-3-modified HS does not bind to AT (Liu et al. (1999) J. Biol.Chem. 274, 5185-5192). As expected, compound 6 does not exhibit anyanti-Xa and antithrombin activities. The binding of AT to thesynthesized compounds was also measured (Table 4). It is clear that theanticoagulant activities of the compounds correlated to their bindingaffinity to AT. Taken together, these results demonstrate that theanticoagulant activities of these enzymatically synthesizedpolysaccharides are consistent with the known structure activityrelationship of HS.

TABLE 4 Anti-factor Xa and antithrombin activities of synthesizedpolysaccharide intermediates Factor Xa inhibition Thrombin inhibitionBinding (IC₅₀)^(a) (IC₅₀)^(a) to AT^(b) Sample ng/ml ng/ml % Heparin^(c)20 10  ND^(d) Heparan sulfate^(e) >5000 >3000 ND Arixtra^(f) 58 >3000 NDCompound 7 >2000 >3000 5 Compound 8 126 96 31 Compound 5 40 32 38Compound 6 >2000 >2000 2 ^(a)The procedures for measuring the activitiesof factor Xa and thrombin are described under “Experimental Procedures.”^(b)3-O—[³⁵S]sulfo compounds were used to determine their bindings to ATas described under “Experimental Procedures.” ³H-Labeled HS from CHOcells was used as a negative control for the AT binding. About 0.3% of³H-labeled HS bound to AT. ^(c)Heparin was from Sigma. ^(d)ND, notdetermined. ^(e)Heparan sulfate was isolated from bovine kidney.^(f)Arixtra is the chemically synthesized antithrombin-bindingpentasaccharide, which was obtained from a local pharmacy.

Preparation of the Polysaccharide that Binds to Herpes Simplex Virus gD.Herpes simplex virus utilizes HS as a receptor to infect the targetcells (Shukla and Spear (2001) J. Clin. Invest, 108, 503-510). Aspecific 3-O-sulfo group-containing HS, generated by 3-OST-3, -5, or -6,serves as an entry receptor for herpes simplex virus type 1 through HSbinding of gD (Shukla et al. (1999) Cell 99, 13-22; Xia et al. (2002) J.Biol. Chem. 277, 37912-37919; Xu et al. (2005) Biochem. J. 385,451-459). Structural characterization of the gD-binding site revealed aunique octasaccharide sequence carrying a 3-O-sulfo glucosamine residue(Liu et al. (2002) J. Biol. Chem. 277, 33456-33467). To test if thepresently disclosed enzymatic approach synthesizes gD-binding HS,compound 4 (FIG. 1A) was incubated with immobilized 3-OST-3 to generatecompound 6 (FIG. 1A). It was found that 12% of the resultantpolysaccharide (compound 6) bound to gD, whereas only 2.6% of compound5, a 3-OST-1-modified polysaccharide, bound to gD as determined byimmunoprecipitation (Table 5). Comparison with the appropriate controlsshowed that the percentage binding for 3-OST-3-modified HS closelyresembled that of compound 6. In conclusion, the results suggest thatthe presently disclosed enzymatic approach is capable of effectivelysynthesizing gD-specific binding HS. It should be noted that commercialheparin does not bind to gD, and heparin is not a substrate for 3-OST-3(Nicola et al. (1996) J. Virol. 70, 3815-3822; Liu et al. (1999) J.Biol. Chem. 274, 38155-38162). Therefore, these results also demonstratethat it is possible to redesign the sulfation patterns by firstsolvolytically removing all O-sulfo groups and then selectivelyenzymatically replacing sulfo groups required for specific interactions.

TABLE 5 The binding of synthesized polysaccharides to gD gD bindingSample % Unmodified HS^(a) 1.0 3-OST-1-modified HS^(b) 2.63-OST-3-modified HS^(b) 12.0 Compound 5 2.6 Compound 6 12.1 ^(a)[³H]HS(from CHO cells) was prepared by metabolically labeling the cells with[³H]glucosamine. ^(b)3-OST-1- and 3-OST-3-modified HS were prepared byincubating [³H]HS with purified 3-OST-1 or 3-OST-3 enzymes.

Discussion of Examples 1-3

HS, including heparin, has a wide range of biological activities,including anticoagulation, antiviral, and anticancer activities. Sulfogroup-containing saccharide sequences dominate the specificity of thefunctions of heparin and HS. Thus, the synthesis of a polysaccharidewith the appropriate positioning of these functional groups to carry outits unique biological activity is desirable. The presently disclosedsubject matter provides an approach to synthesize sulfo group-containingHS polysaccharides that have desired biological activities, includingfor example HS that binds to FGF2, herpes simplex virus gD, or AT. TheseHS polysaccharides can also demonstrate appropriate biological activity,such as anticoagulant activity mediated through AT binding and theactivity in promoting cell proliferation. More importantly, thepresently disclosed subject matter permits the synthesis of greater than1 mg amounts of specific sulfo group-containing polysaccharides,sufficient for testing their activities in biochemical and biologicalassays. The quantities synthesized are also sufficient for extensivestructural analysis, including disaccharide analysis and one- andtwo-dimensional NMR analysis.

In examples 1-3. two approaches were used to increase the scale ofenzymatic synthesis. First, HS sulfotransferases were expressed in E.coli, readily affording 20-50 mg of purified enzymes. It has been shownthat both 3-OST-1 and 3-OST-3 can be expressed in E. coli in relativelyhigh yield. The presently disclosed subject matter also provides for theexpression in E. coli of both 2-OST and 6-OST as the soluble 2-OST and6-OST-MBP fusion proteins. Second, efficiency of enzymatic sulfation wasimproved by utilizing immobilized O-sulfotransferases and coupling theiruse to a PAPS regeneration system.

When a PAPS regeneration system was previously applied for preparingN-sulfoheparosan from heparosan using N-deacetylase/N-sulfotransferase,the N-sulfation yield was lower than that obtained using added exogenousPAPS (Saribas et al. (2004) Glycobiology 14, 1217-1228). Nevertheless,coupling of a PAPS regeneration system to enzymatic sulfation ofpolysaccharides, as disclosed herein, was considered a desirable goalfor several reasons. First, PAP inhibits HS O-sulfotransferaseactivities with IC₅₀ values of ˜100 μM under the reaction conditionsused in the presently disclosed syntheses, making milligram-scalesynthesis difficult without continuously removing PAP. Second, a PAPSregeneration system would permit use of PNPS as the sulfo donor andrequire only catalytic amounts of PAP, significantly reducing the costof synthesis. The listed price of PNPS in the Aldrich catalog is about300-fold less than PAPS. The immobilized enzyme format facilitates useof the enzymes repeatedly, making the method amendable to a large scalesynthesis, since scale-up of immobilized enzyme columns is generallyknown in the art.

Developing an effective approach to synthesize HS is also desirable forunderstanding the mechanism of its biosynthesis. Unlike proteinbiosynthesis, polysaccharide biosynthesis is not a template-drivenprocess. Although the cDNAs encoding the HS biosynthetic enzymes havebeen cloned, the genetic regulation mechanism for the synthesis of theHS with defined biological functions is not fully understood. The uniqueand often remote sequence features in substrate can influence the actionof HS O-sulfotransferases. Furthermore, whether additional factors orthe formation of complexes of biosynthetic enzymes takes part incontrolling the structure of HS remains to be elucidated. It isinteresting to note that a complex of HS epimerase and 2-OST in vivo hasbeen reported (Pinhal et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98,12984-12989). The presently disclosed approach provides a tool to answerthese questions, since it is capable of generating a sufficient amountof HS product required for extensive structural biochemical andbiological analysis.

HS is believed to be present in block structures, comprising highlysulfated and non-sulfated domains (Gallagher, J. T. (2001) J. Clin.Invest 108, 357-361), and the HS with the biological activities largelycontain the highly sulfated domains. Without wishing to be limited bytheory, it is believed that the modifications in the present systemindeed happen in a block fashion based on the following facts. First,the results of the disaccharide analysis concluded that both6-O-sulfation and 2-O-sulfation are carried out predominantly in thesulfated region (FIG. 3 and Table 2). Second, the enzymatically-modifiedproducts exhibit the anticipated biological functions, including theactivation of FGF/FGF receptor signaling (compounds 4a and 4b), carryinganticoagulant activity (compound 5), and binding to herpes simplex virusglycoprotein D (compound 6). Because the HS carrying these functionsmust contain the domain structures with a size larger thanpentasaccharide (Petitou and van Boeckel (2004) Angew. Chem. Int. Ed.43, 3118-3133; Atha et al. (1985) Biochemistry 24, 6723-6729; Liu et al.(2002) J. Biol. Chem. 277, 33456-33467; Maccarana et al. (1993) J. Biol.Chem. 268, 23898-23905), the synthetic products with the desiredfunctions suggest that the modifications indeed occur in a blockfashion.

In summary, the presently disclosed subject matter provides a method forenzymatic sulfation and preparation of HS with distinct biologicalactivities. Unique sulfated saccharide sequences play a dominant role inthe function and specificity of HS/heparin. The presently disclosedmethods demonstrate the capability of using a collection of HSbiosynthetic enzymes to synthesize HS/heparin with selected biologicalactivities. The synthetic scale with this method can be easily increasedfor large scale synthesis, provided that both the enzymes and the sulfodonor are easily accessible. The current method clearly demonstratesthat HS/heparin having specific biological activities can be synthesizedby subjecting a backbone saccharide polymer to different enzymaticmodifications. This enzymatic selectivity is currently not accessible bychemical sulfation approaches. The presently disclosed method cansignificantly aid the exploration of new potential therapeuticapplications for HS. In addition, enzymatic synthesis of anticoagulantheparin can potentially lead to a better anticoagulant drug by reducingits side effects.

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. A method of sulfating a polysaccharide, comprising: (a) providing areaction mixture comprising: at least one O-sulfotransferase (OST)enzyme; and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); (b) incubatinga polysaccharide substrate with the reaction mixture, wherein productionof the sulfated polysaccharide from the polysaccharide substrate iscatalyzed by the OST enzyme with a conversion of the PAPS to adenosine3′,5′-diphosphate (PAP); and (c) providing a reaction condition whichmodifies PAP to reduce an inhibitory effect of PAP on the polysaccharidesulfation.
 2. The method of claim 1, wherein the at least one OST enzymeis selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST,and combinations thereof.
 3. The method of claim 2, wherein the at leastone OST enzyme is a recombinant OST enzyme.
 4. The method of claim 3,wherein the recombinant OST enzyme is produced in a bacterial expressionsystem.
 5. The method of claim 3, wherein the at least one OST enzyme isa fusion protein.
 6. The method of claim 5, wherein the fusion proteinis a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OSTfusion protein.
 7. The method of claim 1, wherein the OST enzyme isimmobilized on a substrate.
 8. The method of claim 7, wherein thesubstrate is an agarose bead.
 9. The method of claim 1, whereinproviding the reaction condition comprises providing a PAPS regenerationsystem comprising a PAPS regenerating enzyme and a sulfur donorcompound, wherein the PAPS regenerating enzyme catalyzes regeneration ofthe PAPS from the PAP utilizing the sulfur donor compound as asubstrate.
 10. The method of claim 9, wherein the PAPS regeneratingenzyme is an arylsulfotransferase.
 11. The method of claim 10, whereinthe arylsulfotransferase is AST-IV.
 12. The method of claim 9, whereinthe sulfur donor compound is an aryl sulfate compound.
 13. The method ofclaim 12, wherein the aryl sulfate compound is p-nitrophenol sulfate(PNPS).
 14. The method of claim 1, wherein providing the reactioncondition comprises providing a phosphatase enzyme, wherein thephosphatase enzyme modifies the PAP.
 15. The method of claim 1, whereinthe polysaccharide substrate is a chemically desulfated N-sulfated(CDSNS) heparin.
 16. The method of claim 1, wherein the polysaccharidesubstrate is partially sulfated prior to reaction mixture incubation.17. The method of claim 1, wherein the sulfated polysaccharide is aglycosaminoglycan (GAG).
 18. The method of claim 17, wherein the GAG isa heparan sulfate (HS).
 19. The method of claim 18, wherein the HS is ananticoagulant-active HS.
 20. The method of claim 18, wherein the HS isselected from the group consisting of an antithrombin-binding HS, afibroblast growth factor (FGF)-binding HS, and a herpes simplex virusenvelope glycoprotein D-binding HS.
 21. A method of sulfating apolysaccharide, comprising: (a) providing a reaction mixture comprisingadenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and asulfur donor compound; (b) incubating the reaction mixture for a timeperiod sufficient to catalyze the production of 3′-phosphoadenosine5′-phosphosulfate (PAPS) from the PAP by the PAPS regenerating enzymeutilizing the sulfur donor compound as a substrate; and (c) incubating apolysaccharide substrate and at least one O-sulfotransferase (OST)enzyme with the reaction mixture, wherein production of a sulfatedpolysaccharide from the polysaccharide substrate is catalyzed by the OSTenzyme with a conversion of the PAPS to PAP and wherein the PAPSregenerating enzyme catalyzes regeneration of the PAPS from the PAPutilizing the sulfur donor compound as a substrate.
 22. The method ofclaim 21, wherein the PAPS regenerating enzyme is anarylsulfotransferase.
 23. The method of claim 22, wherein thearylsulfotransferase is AST-IV.
 24. The method of claim 21, wherein thesulfur donor compound is an aryl sulfate compound.
 25. The method ofclaim 24, wherein the aryl sulfate compound is p-nitrophenol sulfate(PNPS).
 26. The method of claim 21, wherein the time period is fromabout 1 minute to about 30 minutes.
 27. The method of claim 21, whereinthe polysaccharide substrate is a chemically desulfated N-sulfated(CDSNS) heparin.
 28. The method of claim 21, wherein the polysaccharidesubstrate is partially sulfated prior to reaction mixture incubation.29. The method of claim 21, wherein the at least one OST enzyme isselected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST,and combinations thereof.
 30. The method of claim 29, wherein the atleast one OST enzyme is a recombinant OST enzyme.
 31. The method ofclaim 30, wherein the recombinant OST enzyme is produced in a bacterialexpression system.
 32. The method of claim 30, wherein the at least oneOST enzyme is a fusion protein.
 33. The method of claim 32, wherein thefusion protein is a maltose-binding protein (MBP)-2-OST fusion proteinor a MBP-6-OST fusion protein.
 34. The method of claim 21, wherein theOST enzyme is immobilized on a substrate.
 35. The method of claim 34,wherein the substrate is an agarose bead.
 36. The method of claim 21,wherein the sulfated polysaccharide is a glycosaminoglycan (GAG). 37.The method of claim 36, wherein the GAG is a heparan sulfate (HS). 38.The method of claim 37, wherein the HS is an anticoagulant-active HS.39. The method of claim 37, wherein the HS is selected from the groupconsisting of an antithrombin-binding HS, a fibroblast growth factor(FGF)-binding HS, and a herpes simplex virus envelope glycoproteinD-binding HS.
 40. A kit for sulfating a polysaccharide, the kitcomprising: (a) at least one O-sulfotransferase (OST) enzyme; and (b) areagent which modifies adenosine 3′,5′-diphosphate (PAP) to reduce aninhibitory effect of PAP on polysaccharide sulfation.
 41. The kit ofclaim 40, further comprising instructions for sulfating apolysaccharide.
 42. The kit of claim 40, wherein the at least one OSTenzyme is contained within a first container and the reagent iscontained within a second container.
 43. The kit of claim 40, whereinthe at least one OST enzyme is selected from the group consisting of2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof.
 44. The kit ofclaim 43, wherein the at least one OST enzyme is a recombinant OSTenzyme.
 45. The kit of claim 44, wherein the recombinant OST enzyme isproduced in a bacterial expression system.
 46. The kit of claim 44,wherein the at least one OST enzyme is a fusion protein.
 47. The kit ofclaim 46, wherein the fusion protein is a maltose-binding protein(MBP)-2-OST fusion protein or a MBP-6-OST fusion protein.
 48. The kit ofclaim 38, wherein the OST enzyme is immobilized on a substrate.
 49. Thekit of claim 48, wherein the substrate is an agarose bead.
 50. The kitof claim 40, wherein the reagent comprises a PAPS regeneration systemcomprising a PAPS regenerating enzyme and a sulfur donor compound. 51.The kit of claim 50, wherein the PAPS regenerating enzyme is anarylsulfotransferase.
 52. The kit of claim 51, wherein thearylsulfotransferase is AST-IV.
 53. The kit of claim 50, wherein thesulfur donor compound is an aryl sulfate compound.
 54. The kit of claim53, wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS).55. The kit of claim 40, wherein the reagent comprises a phosphataseenzyme.